Methods and compositions for obtaining marker-free transgenic plants

Abstract

The invention provides methods and compositions for identifying transgenic seed that contain a transgene of interest, but lack a marker gene. Use of an identification sequence that results in a detectable phenotype increases the efficiency of screening for seed and plants in which transgene sequences not linked to a gene of interest have segregated from the sequence encoding a gene of interest.

1 - 48 . (canceled) 
     
     
         49 . A method of preparing marker free pollen comprising the steps of:
 (a) obtaining a plant transformation construct comprising (i) a gene of interest; and (ii) a marker gene physically linked to a DNA cassette comprising an identification sequence that is operably linked to a promoter functional in a pollen cell; wherein expression of the DNA cassette affects pollen viability, pollen development, or seed fertilization;   (b) transforming a plant cell with the transformation construct; and obtaining a transgenic plant from the transgenic plant cell.   (c) producing pollen comprising the gene of interest and not comprising the identification sequence.   
     
     
         50 . The method of  claim 49 , further comprising pollinating a second plant with pollen of the transgenic plant; and selecting a seed comprising the gene of interest and not comprising the identification sequence. 
     
     
         51 . The method of  claim 50 , wherein selecting a seed further comprises assaying the seed for the presence of the gene of interest and selecting a seed that comprises the gene of interest. 
     
     
         52 . The method of  claim 49 , wherein the transformation construct is expressed in pollen and seed tissue. 
     
     
         53 . The method of  claim 49 , wherein the DNA cassette comprises DNA encoding an antisense or sense RNA that silences an endogenous gene to result in non-viable pollen. 
     
     
         54 . The method of  claim 53 , wherein the DNA cassette comprises a pair of inverted repeats of a DNA fragment homologous to the endogenous gene. 
     
     
         55 . The method of  claim 54 , wherein the inverted DNA fragment repeat is embedded in an intron within the marker gene. 
     
     
         56 . The method of  claim 49 , wherein the marker gene is a selectable marker gene. 
     
     
         57 . The method of  claim 56 , wherein the selectable marker gene encodes a product selected from the group consisting of CP4 EPSPS, phosphinothricin acetyltransferase, DMO, NptII, glyphosate acetyl transferase, mutant acetolactate synthase, methotrexate resistant DHFR, dalapon dehalogenase, PMI, Protox, hygromycin phosphotransferase and 5-methyl tryptophan resistant anthranilate synthase. 
     
     
         58 . The method of  claim 49 , wherein the gene of interest and the identification sequence are bounded by different T-DNA border sequences. 
     
     
         59 . The method of  claim 49 , wherein the DNA cassette comprises a nucleic acid sequence operably linked to a promoter selected from the group consisting of: an LAT52 promoter, an LAT59 promoter, a maize waxy gene promoter, and a rice small subunit ADP-glucose pyrophosphorylase promoter. 
     
     
         60 . The method of  claim 49 , wherein the identification sequence comprises a first transgene and a second transgene. 
     
     
         61 . The method of  claim 60 , wherein the first transgene of the identification sequence is expressed in pollen, and the second transgene of the identification sequence is expressed in seeds. 
     
     
         62 . The method of  claim 49 , wherein the identification sequence produces a protein lethal to pollen. 
     
     
         63 . The method of  claim 49 , wherein the identification sequence produces a protein that inhibits pollen germination. 
     
     
         64 . A DNA construct comprising (i) a gene of interest; and (ii) a marker gene physically linked to a DNA cassette comprising an identification sequence that is operably linked to a promoter functional in a pollen cell; wherein expression of the DNA cassette affects pollen viability, pollen development, or seed fertilization. 
     
     
         65 . A transgenic cell transformed with the construct of  claim 64 . 
     
     
         66 . The cell of  claim 65 , wherein the cell is a plant cell. 
     
     
         67 . A transgenic plant transformed with the construct of  claim 64 . 
     
     
         68 . A transgenic plant co-transformed with a DNA construct comprising a first DNA segment comprising left and right T-DNA borders flanking a gene of interest operably linked to a promoter functional in plants and a second DNA construct containing a second DNA segment comprising a second set of left and right T-DNA borders flanking a promoter functional in a pollen cell operably linked to a DNA cassette, wherein expression of the DNA cassette affects pollen viability, pollen development or seed fertilization of a plant comprising the DNA cassette, and a marker gene operably linked to a promoter functional in a plant cell. 
     
     
         69 . A cell of the plant of  claim 68 . 
     
     
         70 . A DNA construct comprising right and left T-DNA borders, wherein a first DNA segment comprising a gene of interest operably linked to a promoter functional in plants is located after the left border and a second DNA segment comprising a DNA cassette, wherein expression of the DNA cassette affects pollen viability, pollen development or seed fertilization of a plant comprising the DNA cassette, and a marker gene operably linked to a promoter functional in plants is located after the right border. 
     
     
         71 . A DNA construct comprising right and left T-DNA borders, wherein a first DNA segment comprising a DNA cassette, wherein expression of the DNA cassette affects pollen viability, pollen development or seed fertilization of a plant comprising the DNA cassette and a marker gene operably linked to a promoter functional in plants is located after the right border, and a second DNA segment comprising a gene of interest operably linked to a promoter functional in plants is located after the left border. 
     
     
         72 . A DNA construct comprising first and second right T-DNA borders, wherein a first DNA segment comprising a gene of interest operably linked to a promoter functional in plants is located after the first right border and a second DNA segment comprising a DNA cassette, wherein expression of the DNA cassette affects pollen viability, pollen development or seed fertilization of a plant comprising the DNA cassette, and a marker gene operably linked to a promoter functional in plants is located after the second right border.